Engineered heterodimeric proteins

ABSTRACT

The present invention provides heterodimeric antibodies and fragments thereof and methods for their preparation, wherein the pairing of heavy and light chains has been improved. Interface residues were mutated such that each light chain strongly favoured its cognate heavy chain when two different heavy chains and two different light chains were co-transfected and co-expressed in the same cell to assemble a functional, heterodimeric antibody or fragment thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 20, 2018 isnamed PAT057505-WO-PCT_SL.txt and is 201,737 bytes in size.

FIELD OF THE INVENTION

The invention relates to heterodimeric antibodies and fragments thereofcomprising modifications that promote correct heavy and light chainpairing and methods for their preparation.

BACKGROUND OF THE INVENTION

The administration of heterodimeric antibodies, particularly bispecificantibodies as therapeutic agents for human diseases is of great clinicalpotential but the robust generation of heterodimeric antibodies,especially the production of pure and developable heterodimericantibodies is still challenging. Antibody heavy chains bind antibodylight chains in a promiscuous manner such that a given heavy chain canpair with many light chain sequences of both the lambda and kappa lightchain classes (Edwards B M et al., (2003) J. Mol. Biol. 334:103-18).Previous work has shown that pairing of heavy and light chains occurs atrandom (Brezinschek H P et al., (1998) J. Immunol. 160:4762-7). As aresult of this binding, concomitant expression of two antibody heavychains and two antibody light chains naturally leads to scrambling ofheavy chain-light chain pairings; however homogeneous pairing is anessential requirement for manufacturability and biological efficacy.Heterodimeric antibody formats such as DVD-Ig (Wu C et al., (2007) Nat.Biotech. 25(11):1290-7), CrossMab (Schaefer W et al., (2011) PNAS108(27): 11187-92), or ‘two-in-one’ antibodies (Bostrom J et al., (2009)Science 323(5921): 1610-4) allow the production of bispecific antibodiesbut with varying liabilities. Despite these recent developments therestill exists in the art a need for an improved heterodimeric antibodyformat having the correct heavy and light chain pairing and a method forachieving this homogeneous pairing to avoid the generation of mispairedcontaminants.

SUMMARY OF THE INVENTION

In the present invention, to improve the pairing of heavy and lightchains of heterodimeric antibodies, electrostatic steering mechanismswere applied to engineer the heavy and light chains of a subset ofantibodies and antibody fragments. Interface residues were mutated insuch a way that each light chain strongly favoured its cognate heavychain when two different heavy chains and two different light chainswere co-transfected and co-expressed in the same cell to assemble afunctional, heterodimeric, bispecific antibody.

The design strategy for engineering of heavy chain-light chain pairingincluded identifying a representative Fab. A criterion for arepresentative Fab was that it was a member of commonly used heavy chainvariable domain (VH) and light chain variable domain (VL) subgroup suchas VH3 and Vκ1 or Vλ1. For charge engineering several criteria wereconsidered. The chosen amino acids should not be in contact with thevariable domain CDRs in the conformational structure asinteraction-contacts to CDRs could eventually lead to loss of binding,and thereby should be avoided. The chosen positions should be highlyconserved within most common antibody families, in order to be able togenerate heterodimers of any given subfamily. Additionally, by avoidingpositions in the centre of the interface of the VH with VL, and heavychain constant domain 1 (CH1) with the light chain constant domain (CL),and choosing positions at the rim of this interface, it was possible toachieve an effect that strongly favours heterodimeric formation bycreating salt bridges at the ends of the domains and thereby generatinga “clip-effect” that closes the correct heavy-light chain formation.This “clip-effect” served to stabilize the protein and reducedestabilization of the interface of the constant domains CH1 and CL.Therefore, charged residues at the end of the VH and VL domains and atthe beginning and end of CH1 and CL domains were introduced. Selectionof the residues for charge engineering at the end of the CH1 and CLdomains was based on the observed distance of up to 12 Å between therespective main chains, as opposed to selecting residues formodification based on side chain interactions.

In one aspect, the present invention provides a heterodimeric antibodyor fragment thereof comprising an engineered VH and CH1 domain, and anengineered VL and CL domain comprising a number of substitutions atcertain positions. The numbering of all substitution positions isaccording to EU numbering.

In one embodiment, the present invention provides a heterodimericantibody or fragment thereof comprising an engineered VH domain and CH1domain comprising a charged or neutral amino acid at positions 39, 147and 165; and an engineered VL domain and CL domain comprising a chargedor neutral amino acid at positions 38, 124 and 169/170 (EU numbering),wherein said amino acids in the VH and CH1 domains, and thecorresponding amino acids of the interface in the VL and CL domainspairwise are of opposing charge or are charged/neutral, and form aninterface that is electrostatically favourable to heterodimerization. Toensure that the amino acid at the positions specified is of theappropriate charge (negative, positive or neutral/uncharged), it may benecessary to substitute an amino acid at a specific position in the wildtype sequence with an amino acid of a different charge. For example, theamino acid in the wild type sequence may be neutral, basic or acidic andthe substitution results in a change in charge. More specifically, aneutral amino acid may be substituted with a basic or acidic amino acid,a basic amino acid may be substituted with a neutral or an acidic aminoacid or an acidic amino acid may be substituted with a neutral or abasic amino acid.

In one embodiment, in the VH domain, the neutral amino acid glutamine(Q) at position 39 may be substituted with a basic or acidic amino acid.Examples of basic amino acids (positive charge) include arginine (R),lysine (K) or histidine (H) and examples of acidic amino acids (negativecharge) include aspartic acid (D) and glutamic acid (E). In particular,the substitution is from Q to R, K or D. In the CH1 domain, the basicamino acid K at position 147 may remain unchanged i.e. has a positivecharge or may be substituted with a neutral or an acidic amino acid. Inparticular, the substitution is to an acidic amino acid and is from K toD. In addition, the neutral amino acid serine (S) at position 165 mayremain uncharged i.e. is neutral or may be substituted with a basic oracidic amino acid. In particular the substitution is from S to R or D.In one embodiment, the VH domain is from the VH1, VH2, VH3, VH5 or VH6subfamily. In a particular embodiment, the VH domain is from the VH3subfamily.

In one embodiment, in which the light chain is of the kappa (κ) subtype,in the VL domain the neutral amino acid Q at position 38 may besubstituted with a basic or acidic amino acid. Examples of basic aminoacids include arginine (R), lysine (K) or histidine (H) and examples ofacidic amino acids include aspartic acid (D) and glutamic acid (E). Inparticular, the substitution is from Q to R, K or D. In the CL domain,the neutral amino acid Q at position 124 may remain uncharged i.e. isneutral or may be substituted with a basic or acidic amino acid. Inparticular, the substitution is from Q to K or D. In addition, the basicamino acid K at position 169 may remain unchanged i.e. has a positivecharge or may be substituted with a neutral or an acidic amino acid, inparticular the substitution is to an acidic amino acid and is from K toD. In one embodiment, the kappa light chain is of the Vκ1, or Vκ3subfamily.

In one embodiment in which the light chain is of the lambda (A) subtype,in the VL domain the neutral amino acid Q at position 38 may besubstituted with a basic or acidic amino acid. Examples of basic aminoacids include arginine (R), lysine (K) or histidine (H) and examples ofacidic amino acids include aspartic acid (D) and glutamic acid (E). Inparticular, the substitution is to a basic amino acid and is from Q toK. In the CL domain, the acidic amino acid E at position 124 may besubstituted with a neutral, basic or another acidic amino acid. Inparticular, the substitution is to another acidic amino acid and is fromE to D. In addition, the neutral amino acid N at position 170 may remainuncharged i.e. is neutral or may be substituted with a basic or acidicamino acid. In particular the substitution is to a basic amino acid andis from N to K or is from N to R. In one embodiment, the lambda lightchain is of the Vλ1, Vλ2 or Vλ3 subfamily.

In one embodiment, the engineered VH and CH1 domains may comprisecharged or neutral amino acids at positions 39, 147 and 165 and theengineered VL and CL domains may comprise charged or neutral amino acidsat the corresponding positions 38, 124 and 169/170 of the interface (EUnumbering). As such, the present invention provides a heterodimericantibody or fragment thereof with engineered VH and CH1 domains andengineered VL and CL domains wherein:

-   (i) the engineered VH and CH1 domains comprise an acidic amino acid    at position 39, an acidic amino acid at position 147 and an acidic    amino acid at position 165 and the engineered VL and CL domains    comprise a basic amino acid at position 38, a basic amino acid at    position 124 and a basic amino acid at position 169/170;-   (ii) the engineered VH and CH1 domains comprise a basic amino acid    at position 39, a basic amino acid at position 147 and a basic amino    acid at position 165 and the engineered VL and CL domains comprise    an acidic amino acid at position 38, an acidic amino acid at    position 124 and an acidic amino acid at position 169/170;-   (iii) the engineered VH and CH1 domains may comprise an acidic amino    acid at position 39, an acidic amino acid at position 147 and a    basic amino acid at position 165 and the engineered VL and CL    domains may comprise a basic amino acid at position 38, a basic    amino acid at position 124 and an acidic amino acid at position    169/170;-   (iv) the engineered VH and CH1 domains may comprise a basic amino    acid at position 39, a basic amino acid at position 147 and an    acidic amino acid at position 165 and the engineered VL and CL    domains may comprise an acidic amino acid at position 38, an acidic    amino acid at position 124 and a basic amino acid at position    169/170;-   (v) the engineered VH and CH1 domains may comprise an acidic amino    acid at position 39, a basic amino acid at position 147 and an    acidic amino acid at position 165 and the engineered VL and CL    domains may comprise a basic amino acid at position 38, an acidic    amino acid at position 124 and a basic amino acid at position    169/170;-   (vi) the engineered VH and CH1 domains may comprise a basic amino    acid at position 39, an acidic amino acid at position 147 and a    basic amino acid at position 165 and the engineered VL and CL    domains may comprise an acidic amino acid at position 38, a basic    amino acid at position 124 and an acidic amino acid at position    169/170;-   (vii) the engineered VH and CH1 domains may comprise an acidic amino    acid at position 39, a basic amino acid at position 147 and an    acidic amino acid at position 165 and the engineered VL and CL    domains may comprise a basic amino acid at position 38, an acidic    amino acid at position 124 and a neutral amino acid at position    169/170;-   (viii) the engineered VH and CH1 domains may comprise a basic amino    acid at position 39, an acidic amino acid at position 147 and a    neutral amino acid at position 165 and the engineered VL and CL    domains may comprise an acidic amino acid at position 38, a basic    amino acid at position 124 and a basic amino acid at position    169/170; or-   (ix) the engineered VH and CH1 domains may comprise an acidic amino    acid at position 39, a basic amino acid at position 147 and a    neutral amino acid at position 165 and the engineered VL and CL    domains may comprise a basic amino acid at position 38, a neutral    amino acid at position 124 and a basic amino acid at position    169/170.

In one embodiment, the engineered VH and CH1 domains may comprise abasic amino acid at position 39, an acidic amino acid at position 147and a neutral amino acid at position 165 and the engineered VL and CLdomains may comprise an acidic amino acid at position 38, a basic aminoacid at position 124 and a basic amino acid at position 169 (EUnumbering). Alternatively, the engineered VH and CH1 domains maycomprise an acidic amino acid at position 39, a basic amino acid atposition 147 and a neutral amino acid at position 165 and the engineeredVL and CL domains may comprise a basic amino acid at position 38, aneutral amino acid at position 124 and a basic amino acid at position169 (EU numbering). Alternatively, the engineered VH and CH1 domains maycomprise an acidic amino acid at position 39, a basic amino acid atposition 147 and an acidic amino acid at position 165 and the engineeredVL and CL domains may comprise a basic amino acid at position 38, anacidic amino acid at position 124 and a neutral amino acid at position1709 (EU numbering).

In a specific embodiment, the present invention provides a heterodimericantibody or fragment thereof having engineered VH and CH1 domainscomprising at positions 39, 147 and 165 (EU numbering) an acidic aminoacid, a basic amino acid and an acidic amino acid, respectivelytherefore resulting in a negative, positive and negative charge,respectively at these positions, or vice versa. The correspondingengineered VL and CL domains comprise at positions 38, 124 and 169/170(EU numbering) an amino acid having an opposing charge to that of thecorresponding heavy chain interface amino acid. For example, if theengineered heavy chain comprises at position 39 an acidic amino acid, atposition 147 a basic amino acid and at position 165 an acidic aminoacid, then the corresponding light chain will comprise at position 38 abasic amino acid, at position 124 an acidic amino acid and at position169/170 a basic amino acid (EU numbering). Alternatively, if theengineered heavy chain comprises at position 39 a basic amino acid, atposition 147 an acidic amino acid and at position a basic amino acid,then the corresponding light chain will comprise at position 38 anacidic amino acid, at position 124 a basic amino acid and at position169/170 an acidic amino acid (EU numbering).

In one embodiment, the present invention provides a heterodimericantibody or fragment thereof comprising substitutions selected from thegroup consisting of:

-   i. Q39D, S165D in the VH and CH1, and Q38K in the VL,-   ii. Q39K, K147D in the VH and CH1, and Q38D, Q124K in the kappa VL    and CL;-   iii. Q39D, S165D in the VH and CH1, and Q38K, Q124D in the kappa VL    and CL;-   iv. Q39K, K147D, S165R in the VH and CH1, and Q38D, Q124K, K169D in    the kappa VL and CL;-   v. Q39D, S165D in the VH and CH1, and Q38K, E124D, N170K in the    lambda VL and CL-   vi. Q39D, S165D in the VH and CH1, and Q38K, N170R in the lambda VL    and CL.

In one embodiment, the present invention provides a heterodimericantibody or fragment thereof comprising at least two Fabs, wherein eachFab binds to a different epitope on an antigen. The epitopes may be onthe same antigen or alternatively, the epitopes may be on differentantigens. In one embodiment the heterodimeric antibody or fragmentthereof may be multispecific such as bispecific or trispecific. In sucha multispecific antibody, the amino acids at the interface of the VH andCH1 with the corresponding VL and CL need to be of opposing charge orcharge/neutral, to ensure that an interface is formed that iselectrostatically favourable to heterodimerization. For example, in abispecific antibody or fragment thereof, a Fab that binds to a firstepitope may have engineered VH and CH1 domains comprising at positions39, 147 and 165 (EU numbering) an acidic amino acid, a basic amino acidand an acidic amino acid, respectively, and the corresponding engineeredVL and CL domains comprise at positions 38, 124 and 169/170 (EUnumbering) an amino acid having an opposing charge to that of thecorresponding heavy chain interface amino acid. The Fab that binds to asecond epitope may therefore have engineered VH and CH1 domainscomprising at positions 39, 147 and 165 (EU numbering) a basic aminoacid, an acidic amino acid and a basic amino acid, respectively, and thecorresponding engineered VL and CL domains comprise at positions 38, 124and 169/170 (EU numbering) an amino acid having an opposing charge tothat of the corresponding heavy chain interface amino acid. Therefore,the engineered light chains will only associate with their cognate heavychain of corresponding charge to ensure correct heavy chain-light chainpairing.

In a specific embodiment, the present invention provides a bispecificantibody or fragment thereof, comprising a first Fab comprisingengineered VH and CH1 domains comprising the substitutions Q39K, K147Dand S165R and engineered VL and CL domains comprising the substitutionsQ38D, Q124K and K169D, and a second Fab comprising engineered VH and CH1domains comprising the substitutions Q39D and S165D and engineered kappaVL and CL domains comprising the substitutions Q38K and Q124D, orengineered lambda VL and CL domains comprising the substitutions Q38K,Q38K and N170R or Q38K, E124D and N170K.

In a further aspect, the present invention provides a heterodimericantibody or fragment thereof comprising a Fc region. In one embodiment,the Fc region comprises engineered CH3 domains comprising modificationsaccording to the “knobs-into-holes” approach as described in, e.g.,Ridgway J B B et al., (1996) Protein Engineering, 9(7): 617-21 and U.S.Pat. No. 5,731,168. This approach has been shown to promote theformation of heterodimers of heavy chains and hinder the assembly ofcorresponding heavy chain homodimers. In this approach, a knob iscreated by replacing small amino side chains at the interface betweenCH3 domains with larger ones, whereas a hole is constructed by replacinglarge side chains with smaller ones. In one embodiment, the “knob”mutation comprises T366W and the “hole” mutations comprise T366S, L368Aand Y407V (Atwell S et al., (1997) J. Mol. Biol. 270: 26-35). In aspecific embodiment, the “knob” mutations comprise T366W, S354C and the“hole” mutations comprise T366S, L368A, Y407V and Y349C, so that adisulphide bond is formed between the corresponding cysteine residuesS354C and Y349C, further promoting heterodimer formation.

Another aspect of the present invention provides a method of preparingan heterodimeric antibody or fragment thereof comprising an engineeredVH and CH1 domain and an engineered VL and CL domain, the methodcomprising substituting at least two amino acids in the VH and CH1domains such that the engineered domains comprise a charged or neutralamino acid at positions 39, 147 and 165 (EU numbering), and substitutingan amino acid in the VL and CL domains such that the engineered domainscomprise charged or neutral amino acids at positions 38, 124 and 169/170(EU numbering), and wherein said amino acids in the VH and CH1 domainsand the corresponding amino acids of the interface in the VL and CLdomains pairwise are of opposing charge or are charged/neutral and forman interface that is electrostatically favourable to heterodimerization.

In yet another aspect of the present invention, the electrostaticsteering mechanisms as described herein, that promote correct heavy andlight chain pairing for heterodimeric antibodies and fragments thereof,were combined with modifications to VH-VL and CH1-CL interface of theheterodimeric antibody to engineer an interchain disulfide bond. Thisinterchain disulfide bond further promotes cognate heavy and light chainpairing. In addition, the engineered disulfide bond facilitates theanalytical procedure for profiling the heterodimeric antibodiespermitting easy identification and quantification of misassembledmolecules by a simple electrophoresis based procedure. This results in arobust, high-throughput platform for screening of heterodimericantibodies and fragments thereof with correct heavy chain-light chainpairing.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows a SDS-polyacrylamide gel subjected to electrophoresis undernon-reducing conditions, in which purified bispecific antibodies havebeen loaded. Details of the wells with the corresponding bispecificantibody arm modifications are shown in Table 9. In wells 2 to 5, theknob arm of the bispecific antibody comprised modifications relating tocharge engineering and the hole arm comprised modification related tocharge engineering plus disulfide bond engineering. In wells 7 to 10,the knob and hole arms comprise no modifications relating to chargeengineering but the hole arm comprises modifications relating todisulfide bond engineering. As can be observed for wells 7 to 10 incomparison to wells 2 to 5, disulfide bond engineering alone wasinsufficient to achieve correct heavy chain-light chain pairing. Rows 1,6 and 11 show the molecular weight marker.

DETAILED DESCRIPTION

Disclosed herein are heterodimeric antibodies or fragments thereofcomprising mutated heavy and light chains that have a high degree ofheterodimerization. Also disclosed herein are methods that utilizeelectrostatic steering to select interface residues which when mutated,result in an increase in the correct paring of a light chain with itscognate heavy chain. Co-transfection and co-expression of the mutatedheavy and light chains in the same cell gives rise to the assembly of afunctional, heterodimeric antibody or antibody fragment.

Definitions

As used herein, the term “antibody” refers to a protein, e.g., animmunoglobulin chain or fragment thereof, comprising at least oneimmunoglobulin variable domain sequence. The term “antibody” includes,for example, a monoclonal antibody (including a full length antibodywhich has an immunoglobulin Fc region). In an embodiment, an antibodycomprises a full length antibody, or a full length immunoglobulin chain.In an embodiment, an antibody comprises an antigen binding or functionalfragment of a full length antibody, or a full length immunoglobulinchain.

In an embodiment, an antibody can include a heavy (H) chain variabledomain sequence (abbreviated herein as VH), and a light (L) chainvariable domain sequence (abbreviated herein as VL). In an embodiment,an antibody comprises or consists of a heavy chain and a light chain(referred to herein as a half antibody). In another example, an antibodyincludes two VH sequences and two VL sequences, thereby forming twoantigen binding sites, such as Fab, Fab′, F(ab′)2, Fc, Fd, Fd′, Fv,single chain antibodies (scFv for example), single variable domainantibodies, diabodies (Dab) (bivalent and bispecific), and chimeric(e.g., humanized) antibodies, which may be produced by the modificationof whole antibodies or those synthesized de novo using recombinant DNAtechnologies. These functional antibody fragments retain the ability toselectively bind with their respective antigen or receptor. Antibodiesand antibody fragments can be from any class of antibodies including,but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass(e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies. The preparation of anantibody can be monoclonal or polyclonal. An antibody can also be ahuman, humanized, CDR-grafted, or in vitro generated antibody. Theantibody can have a heavy chain constant region chosen from, e.g., IgG1,IgG2, IgG3, or IgG4. The antibody can also have a light chain chosenfrom, e.g., kappa or lambda. The term “immunoglobulin” (Ig) is usedinterchangeably with the term “antibody” herein.

In an embodiment, an antibody comprises an antigen-binding fragment ofan antibody. Examples of such fragments include: (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a diabody(dAb) fragment, which consists of a VH domain; (vi) a camelid orcamelized variable domain; (vii) a single chain Fv (scFv), see e.g.,Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) PNASUSA 85:5879-5883); (viii) a single domain antibody. These antibodyfragments are obtained using conventional techniques known to those withskill in the art, and the fragments are screened for utility in the samemanner as are intact antibodies.

A Fab as used herein refers to a polypeptide that comprises a VH, CH1,VL and CL immunoglobulin domain. Fab may refer to this polypeptideregion in isolation, or as a polypeptide in the context of a full lengthantibody or antibody fragment.

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence which can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may or may not include one, two, or more N- orC-terminal amino acids, or may include other alterations that arecompatible with formation of the protein structure.

The term “antigen-binding site” refers to the part of an antibody thatcomprises determinants that form an interface that binds to the antigen,or an epitope thereof. With respect to proteins (or protein mimetics),the antigen-binding site typically includes one or more loops (of atleast four amino acids or amino acid mimics) that form an interface thatbinds to the antigen polypeptide. Typically, the antigen-binding site ofan antibody molecule includes at least one or two CDRs and/orhypervariable loops, or more typically at least three, four, five or sixCDRs and/or hypervariable loops.

In one embodiment, the antibody can be recombinantly produced, e.g.,produced by phage display or by combinatorial methods. Phage display andcombinatorial methods for generating antibodies are known in the art (asdescribed in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.,WO 92/18619; Dower et al., WO 91/17271; Winter et al., WO 92/20791;Markland et al., WO 92/15679; Breitling et al., WO 93/01288; McCaffertyet al., WO 92/01047; Garrard et al., WO 92/09690; Ladner et al., WO90/02809; Fuchs et al., (1991) Bio/Technology 9:1370-1372; Hay et al.,(1992) Hum Antibody Hybridomas 3:81-85; Huse et al., (1989) Science246:1275-1281; Griffths et al., (1993) EMBO J 12:725-734; Hawkins etal., (1992) J Mol Biol 226:889-896; Clackson et al., (1991) Nature352:624-628; Gram et al., (1992) PNAS 89:3576-3580; Garrard et al.,(1991) Bio/Technology 9:1373-1377; Hoogenboom et al., (1991) Nuc AcidRes 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982, thecontents of all of which are incorporated by reference herein).

In one embodiment, the antibody is a fully human antibody (e.g., anantibody made in a mouse which has been genetically engineered toproduce an antibody from a human immunoglobulin sequence or an antibodyisolated from a human), or a non-human antibody, e.g., a rodent (mouseor rat), goat, primate (e.g., monkey), camel antibody. Human monoclonalantibodies can be generated using transgenic mice carrying the humanimmunoglobulin genes rather than the mouse system. Splenocytes fromthese transgenic mice immunized with the antigen of interest are used toproduce hybridomas that secrete human monoclonal antibodies withspecific affinities for epitopes from a human protein (see, e.g., Woodet al., WO 91/00906, Kucherlapati et al., WO 91/10741; Lonberg et al.,WO 92/03918; Kay et al., WO 92/03917; Lonberg, N. et al., (1994) Nature368:856-859; Green, L. L. et al., (1994) Nature Genet. 7:13-21;Morrison, S. L. et al., (1994) PNAS USA 81:6851-6855; Bruggeman et al.,(1993) Year Immunol 7:33-40; Tuaillon et al., (1993) PNAS 90:3720-3724;Bruggeman et al., (1991) Eur J Immunol 21:1323-1326).

An antibody can be one in which the variable region, or a portionthereof, e.g., the CDRs, are generated in a non-human organism, e.g., arat or mouse. Chimeric, CDR-grafted, and humanized antibodies are withinthe invention. Antibodies generated in a non-human organism, e.g., a rator mouse, and then modified, e.g., in the variable framework or constantregion, to decrease antigenicity in a human are within the invention.Chimeric antibodies can be produced by recombinant DNA techniques knownin the art (see Robinson et al., WO 87/002671; Akira et al., EP184187A1;Taniguchi, M., EP171496A1; Morrison et al., EP173494A1; Neuberger etal., WO 86/01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly etal., EP125023A1; Better et al., (1988) Science 240:1041-1043; Liu etal., (1987) PNAS 84:3439-3443; Liu et al., (1987), J. Immunol.139:3521-3526; Sun et al., (1987) PNAS 84:214-218; Nishimura et al.,(1987), Canc. Res. 47:999-1005; Wood et al., (1985) Nature 314:446-449;and Shaw et al., (1988), J. Natl Cancer Inst. 80:1553-1559).

A humanized or CDR-grafted antibody will have at least one or two butgenerally all three recipient CDRs (of heavy and or light immunoglobulinchains) replaced with a donor CDR. The antibody may be replaced with atleast a portion of a non-human CDR or only some of the CDRs may bereplaced with non-human CDRs. It is only necessary to replace the numberof CDRs required for binding of the humanized antibody to the targetantigen. Preferably, the donor will be a rodent antibody, e.g., a rat ormouse antibody, and the recipient will be a human framework or a humanconsensus framework. Typically, the immunoglobulin providing the CDRs iscalled the ‘donor’ and the immunoglobulin providing the framework iscalled the ‘acceptor’. In one embodiment, the donor immunoglobulin is anon-human (e.g., rodent). The acceptor framework is anaturally-occurring (e.g., a human) framework or a consensus framework,or a sequence about 85% or higher, preferably 90%, 95%, 99% or higheridentical thereto.

As used herein, the term ‘consensus sequence’ refers to the sequenceformed from the most frequently occurring amino acids (or nucleotides)in a family of related sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family ofproteins, each position in the consensus sequence is occupied by theamino acid occurring most frequently at that position in the family. Iftwo amino acids occur equally frequently, either can be included in theconsensus sequence. A ‘consensus framework’ refers to the frameworkregion in the consensus immunoglobulin sequence.

An antibody can be humanized by methods known in the art (see e.g.,Morrison, S. L., (1985), Science 229:1202-1207; Oi et al., (1986),BioTechniques 4:214, and Queen et al., U.S. Pat. Nos. 5,585,089,5,693,761 and 5,693,762, the contents of all of which are herebyincorporated by reference). Humanized or CDR-grafted antibodies can beproduced by CDR-grafting or CDR substitution, wherein one, two, or allCDRs of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No.5,225,539; Jones et al., (1986) Nature 321:552-525; Verhoeyan et al.,(1988) Science 239:1534; Beidler et al., (1988) J. Immunol.141:4053-4060 and Winter U.S. Pat. No. 5,225,539, the contents of all ofwhich are hereby expressly incorporated by reference. Also within thescope of the invention are humanized antibodies in which specific aminoacids have been substituted, deleted or added. Criteria for selectingamino acids from the donor are described in U.S. Pat. No. 5,585,089,e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of whichare hereby incorporated by reference. Other techniques for humanizingantibodies are described in Padlan et al., EP 519596 A1.

In yet other embodiments, the antibody has a heavy chain constant regionchosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3,IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from, e.g.,the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, andIgG4. In another embodiment, the antibody molecule has a light chainconstant region chosen from, e.g., the (e.g., human) light chainconstant regions of kappa or lambda. The constant region can be altered,e.g., mutated, to modify the properties of the antibody (e.g., toincrease or decrease one or more of: Fc receptor binding, antibodyglycosylation, the number of cysteine residues, effector cell function,and/or complement function). In one embodiment, the antibody haseffector function and can fix complement. In other embodiments, theantibody does not recruit effector cells or fix complement. In anotherembodiment, the antibody has reduced or no ability to bind an Fcreceptor. For example, it is a subtype, isotype, fragment or othermutant, which does not support binding to an Fc receptor, e.g., it has amutagenized or deleted Fc receptor binding region.

Methods for altering an antibody constant region are known in the art.Antibodies with altered function, e.g. altered affinity for an effectorligand, such as FcR on a cell, or the C1 component of complement can beproduced by replacing at least one amino acid residue in the constantportion of the antibody with a different residue (see e.g., EP388151A1,U.S. Pat. Nos. 5,624,821 and 5,648,260, the contents of all of which arehereby incorporated by reference). Similar type of alterations could bedescribed which if applied to the murine, or other speciesimmunoglobulin would reduce or eliminate these functions.

A ‘conservative amino acid substitution’ is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine (K), arginine (R), histidine (H)), acidic sidechains (e.g., aspartic acid (D), glutamic acid (E)), uncharged polarside chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine(S), threonine (T), tyrosine (Y), cysteine (C)), nonpolar side chains(e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline(P), phenylalanine (F), methionine (M), tryptophan (VV)), beta-branchedside chains (e.g., threonine (T), valine (V), isoleucine (I)) andaromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan(VV), histidine (H)).

Heterodimeric Antibodies

In an embodiment, an antibody is a heterodimeric antibody, e.g., itcomprises a plurality of immunoglobulin variable domains sequences,wherein a first immunoglobulin variable domain sequence of the pluralityhas binding specificity for a first epitope and a second immunoglobulinvariable domain sequence of the plurality has binding specificity for asecond epitope. In an embodiment, the first and second epitopes are onthe same antigen, e.g., the same protein (or subunit of a multimericprotein). In another embodiment, the first and second epitopes are ondifferent antigens, e.g., the different proteins (or different subunitsof a multimeric protein). In an embodiment, a heterodimeric antibodycomprises a third, fourth or fifth immunoglobulin variable domain. In anembodiment, a heterodimeric antibody is a bispecific antibody, atrispecific antibody or tetraspecific antibody.

In a specific embodiment, the heterodimeric antibody is a bispecificantibody or fragment thereof. A bispecific antibody has specificity forno more than two epitopes. A bispecific antibody is characterized by afirst immunoglobulin variable domain sequence which has bindingspecificity for a first epitope and a second immunoglobulin variabledomain sequence that has binding specificity for a second epitope. In anembodiment, the first and second epitopes are on the same antigen, e.g.,the same protein (or subunit of a multimeric protein). In an embodiment,the first and second epitopes overlap. In an embodiment, the first andsecond epitopes do not overlap. In an embodiment, the first and secondepitopes are on different antigens, e.g., the different proteins (ordifferent subunits of a multimeric protein). In an embodiment, abispecific antibody comprises a heavy chain variable domain sequence anda light chain variable domain sequence which have binding specificityfor a first epitope and a heavy chain variable domain sequence and alight chain variable domain sequence which have binding specificity fora second epitope. In an embodiment, a bispecific antibody moleculecomprises a half antibody having binding specificity for a first epitopeand a half antibody having binding specificity for a second epitope. Inan embodiment, a bispecific antibody molecule comprises a half antibody,or fragment thereof, having binding specificity for a first epitope anda half antibody, or fragment thereof, having binding specificity for asecond epitope. In an embodiment, a bispecific antibody or fragmentthereof comprises a Fab having binding specificity for a first epitopeand a Fab having binding specificity for a second epitope.

The most commonly used method for the production of heterodimericantibodies, particularly bispecific antibodies is by separate expressionof the component antibodies in different host cells, followed bypurification and assembly into a functional IgG. However, such a methodis costly and involves complex manufacturing processes. Therefore,single host cell expression systems are preferable but have beenassociated with issues of heavy chain and light chain pairing problems.

The heavy chain pairing problem reflects the ability of heavy chains toform homodimers as well as heterodimers when expressed in a single hostcell. The homodimerization of the two heavy chains in an IgG is mediatedby the interaction between the CH3 domains. One of the initial methodsgenerated to improve the heterodimerization of heavy chains utilized the“knobs-into-holes” strategy (Ridgway et al., (1996) supra). Additionalmethods to improve heavy chain heterodimerization include inter alia therational design of electrostatic steering mutations (Gunasekaran et al.,(2010) JBC, 285: 19637-46; Strop et al., (2012) J. Mol. Biol. 420:204-19), the use of computational design (Moore et al., (2011) mAbs, 3:546-557; Von Kreudenstein et al., (2013) mAbs 5: 646-54), theexploitation of sequence divergence but structural similarity of the CH3domains of IgG and IgA (Strand Exchange Engineered Domains (SEED)platform; Davis et al., (2010) Protein Eng. Des. Sel., 23: 195-202) andthe use of a common heavy chain (Fischer et al., (2015) Nat Commun. 6:6113). Recent work described in WO 16/118742 (Eli Lilly and Co), relatesto bispecific antibodies that comprise substitutions in both CH3 domainsto achieve heavy chain heterodimerization. These substitutions were alsocombined with substitutions to the VH and VL domains and CH1 and CLdomains as described in Lewis et al., (2014) Nat. Biotech. 32: 191-8 andWO 14/150973 (Eli Lilly & Co; discussed below).

To address the light chain-heavy chain mispairing problem, previousmethods have included the generation of bispecific antibodies using asingle light chain. This requires heavy-light chain engineering or novelantibody libraries that utilize a single light chain that limits thediversity (Merchant et al., (1998) Nat. Biotech. 16(7):677-81). Inaddition, antibodies with a common light chain have been identified fromtransgenic mice with a single light chain (WO 11/097603 Regeneron;Dhimolea & Reichert (2012) mAbs, 4: 4-13). Another approach is to swapthe CH1 domain of one heavy chain with CL domain of its cognate lightchain (Crossmab technology, Schaefer et al., (2011) supra), which canalso include the “knobs-into-holes” method to ensure heavy chainheterodimerization (Merchant et al., (1998) supra). Design of anorthogonal CH1-CL interface is also possible (Lewis S M et al., (2014)supra) or the use of electrostatic steering mechanisms to engineerantibody light chain-heavy chain interface residues (Liu Z et al.,(2015) JBC, 290:7535-62). Analysis of Fab interfaces revealed thathydrogen bonds and Van-der-Waals interactions are dominant, whereaselectrostatic interactions are rare between the light chain and heavychain.

Additional protocols for generating heterodimeric antibodies include,but not limited to, for example: the electrostatic steering Fc pairingas described in, e.g., WO 09/089004, WO 06/106905 and WO 10/129304; Fabarm exchange as described in, e.g., WO 08/119353, WO 11/131746, and WO13/060867; double antibody conjugate, e.g., by antibody cross-linking togenerate a bi-specific structure using a heterobifunctional reagenthaving an amine-reactive group and a sulfhydryl reactive group asdescribed in, e.g., U.S. Pat. No. 4,433,059; bispecific antibodydeterminants generated by recombining half antibodies (heavy-light chainpairs or Fabs) from different antibodies through cycle of reduction andoxidation of disulfide bonds between the two heavy chains, as describedin, e.g., U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., threeFab′ fragments cross-linked through sulfhydryl reactive groups, asdescribed in, e.g., U.S. Pat. No. 5,273,743; biosynthetic bindingproteins, e.g., pair of scFvs cross-linked through C-terminal tailspreferably through disulfide or amine-reactive chemical cross-linking,as described in, e.g., U.S. Pat. No. 5,534,254; bifunctional antibodies,e.g., Fab fragments with different binding specificities dimerizedthrough leucine zippers (e.g., c-fos and c-jun) that have replaced theconstant domain, as described in, e.g., U.S. Pat. No. 5,582,996;bispecific and oligospecific mono- and oligovalent receptors, e.g.,VH-CH1 regions of two antibodies (two Fab fragments) linked through apolypeptide spacer between the CH1 region of one antibody and the VHregion of the other antibody typically with associated light chains, asdescribed in, e.g., U.S. Pat. No. 5,591,828; bispecific DNA-antibodyconjugates, e.g., crosslinking of antibodies or Fab fragments through adouble stranded piece of DNA, as described in, e.g., U.S. Pat. No.5,635,602; bispecific fusion proteins, e.g., an expression constructcontaining two scFvs with a hydrophilic helical peptide linker betweenthem and a full constant region, as described in, e.g., U.S. Pat. No.5,637,481; multivalent and multispecific binding proteins, e.g., dimerof polypeptides having first domain with binding region of Ig heavychain variable region, and second domain with binding region of Ig lightchain variable region, generally termed diabodies (higher orderstructures are also disclosed creating bispecific, trispecific, ortetraspecific molecules, as described in, e.g., U.S. Pat. No. 5,837,242;minibody constructs with linked VL and VH chains further connected withpeptide spacers to an antibody hinge region and CH3 region, which can bedimerized to form bispecific/multivalent molecules, as described in,e.g., U.S. Pat. No. 5,837,821; VH and VL domains linked with a shortpeptide linker (e.g., 5 or 10 amino acids) or no linker at all in eitherorientation, which can form dimers to form bispecific diabodies; trimersand tetramers, as described in, e.g., U.S. Pat. No. 5,844,094; String ofVH domains (or VL domains in family members) connected by peptidelinkages with cross linkable groups at the C-terminus further associatedwith VL domains to form a series of FVs (or scFvs), as described in,e.g., U.S. Pat. No. 5,864,019; and single chain binding polypeptideswith both a VH and a VL domain linked through a peptide linker arecombined into multivalent structures through non-covalent or chemicalcrosslinking to form, e.g., homobivalent, heterobivalent, trivalent, andtetravalent structures using both scFv or diabody type format, asdescribed in, e.g., U.S. Pat. No. 5,869,620.

Charge Engineering

Specific examples of the use of charge engineering to generatebispecific antibody molecules include SEED heterodimer formation asdescribed in, e.g., WO 07/110205 (Merck) as well as the technologiesdescribed in WO 09/089004 (Amgen), EP1870459B1 (Chugai), WO 10/129304(Oncomed), WO 14/150973 (Eli Lilly & Co and University of NorthCarolina) and WO 16/118742 (Eli Lilly and & Co).

The SEED heterodimers as described in, WO 07/110205 (Merck), compriseengineered CH3 domains of IgA and IgG, wherein the first and secondengineered domains form heterodimers with one another preferentiallyover forming homodimers. In addition, the Fc region can bedifferentially tagged to exploit the inability of human IgG3 isotype tobind to protein A enabling the efficient purification of heterodimers(U.S. Pat. No. 8,586,713).

WO 09/089004 (Amgen Inc.) describes and exemplifies methods for makingbispecific antibody molecules using charge engineering in the CH3 domainto electrostatically favour heavy chain heterodimer formation overhomodimer formation. This application further suggests that to increasethe fidelity of each light chain to bind to the proper heavy chain, theCH1 domain of the heavy chains and constant region of the light chaincan also be engineered to favour dimerization. Their analysis of thelight chain-heavy chain interaction revealed positions in which chargepairs could be introduced into the sequence to enhance binding of aspecific light and heavy chain pair. These positions included T112 oflambda light chain and A141 of the heavy chain, E156 of lambda lightchain and S176 of the heavy chain, and S171 of lambda light chain andS183 of the heavy chain. Additional positions were shown in bold type inTables 4 and 5 of WO 09/089004.

EP1870459B1 (Chugai) reports that the association between VH and VL canbe regulated by substituting amino acids present at the VH-VL interfacewith charged amino acids, which is more effective at formingheterologous molecules than ‘knob into hole’ technology. They suggestthat this can be applied not only to the regulation of associationbetween VH and VL, but can also be applied to the regulation ofassociations among arbitrary polypeptides. Suggested modifications arein the VH/VL interface of an sc(Fv)2. Preparations of VH/VL interfacemodified sc(Fv)2 included modifications at Q39 of the VH and Q38 of theVL, and P44 of the VL.

In WO 10/129304 (Oncomed Pharmaceuticals. Inc.), methods are describedin which altered electrostatic and/or hydrophobic/hydrophilicinteractions between polypeptides in heterodimeric molecules favours theformation of heavy chain heteromultimers over homomultimers. Amino acidsthat interact at the interface between two polypeptides were selectedfor modification. An amino acid residue involved in hydrophilicinteractions was replaced with a more hydrophobic amino acid residueand/or an amino acid involved in a charge interaction with another aminoacid. Positions 236, 245, 249, 278, 286, and 288 in the CH3 domain ofhuman IgG2 were selected for substitutions.

Work described in WO 14/150973 (Eli Lilly & Co and University of NorthCarolina), relates to Fabs and bispecific antibodies with designedresidues in the interfaces of the VH and VL domains and CH1 and CLdomains. Substitutions were made at position 62 in the VH and position 1in the VL and/or position 39 in the VH and position 38 in the VL (Kabatnumbering). Further substitutions were made at positions 172 and/or 174in the CH1 and positions 135 and/or 176 in the CL. A further ‘chargeswop’ substitution of K228D in the heavy chain and D122k in the lightchain was also made to try and improve correct heavy chain-light chainpairing. Even with various combinations of these substitutions thepercentage of correct heavy chain-light chain pairing that could beachieved was still below 90%.

More recent work described in WO 16/118742 (Eli Lilly & Co), relates tobispecific antibodies that comprise substitutions in both CH3 domainsbased on computational and rational design, to improve heavy chainheterodimerization, at inter alia positions: 407 with 366, 409; 407, 399with 366, 409; 360, 399, 407 with 345, 347, 366, 409; 349, 370 with 357,364; and 349, 366, 370, 409 with 357, 364, 407. In addition, mutationswere made to the VH and VL domains as described in Lewis et al., (2014)supra and WO 14/150973 as described in detail above. Only with theseadditional CH3 mutations could an improvement in heavy chain-light chainbinding be achieved over that observed in WO 14/50973.

Whilst the concept of electrostatic engineering has also been used inthe present invention, the inventors have applied specific criteria tothe selection of positions for charge modification. Choosing positionsat the rim of the interface of the heavy chain-light chain pairing,rather than at the centre of the interaction achieves a ‘clip-effect’favouring correct heavy chain-light chain pairing by aligning thecorrect heavy-light chain formation via strong salt-bridges.Substituting charged residues (acidic or basic, where appropriate) atthe end of the variable domains and at the beginning and end of the CH1domain and CL region also reduces the destabilisation of the interfaceof the CH1 and CL. Such modifications have resulted in achieving acorrect paring of heavy chain with its cognate light chain of up to 100%and only minimal destabilisation of the interacting domains.

Disulfide Bond Engineering

Previous work has described that an enhancement of cognate LC and HCpairing can be achieved by the replacement of a native interchaindisulfide bond within one CH1-CL interface with an engineered interchaindisulfide bond (US20140348839 (MedImmune)). In an embodiment of thepresent invention, the combination of charge engineering and disulfidebond engineering has been applied. The native interchain disulfide bondwas replaced with a bond within the VH-VL interface using a design basedon the Fv stabilizing VH44-VL100 disulfide bond widely described in theliterature (Reiter et al., (1996) Nat. Biotech. 14: 1239-45; Weatherillet al., (2012) Protein Eng. Des. Sel. 25(7): 321-9). Such a modificationis intended to covalently lock the desired specific HC/LC pairing and inaddition facilitates the analytical procedure needed to profile thepreparations of bispecific antibodies. Misassembled molecules can beeasily identified and quantified using a simple electrophoresis basedprocedure which further adds to the stringency of the electrostaticengineering method described herein to achieve correctly pairedbispecific antibodies. With the addition of disulfide bond engineeringto heavy and light chain pairs already modified by electrostaticengineering but which had not shown 100% correct cognate pairing,misassembled molecules could be eliminated resulting in bispecificantibodies having a correct paring of heavy chain with their cognatelight chain of up to 100%.

Nucleic Acids and Expression Systems

The present invention also encompasses nucleic acids encoding the heavyand/or light chain constant and/or variable domains described herein.Nucleic acid molecules of the invention include DNA and RNA in bothsingle-stranded and double-stranded form, as well as the correspondingcomplementary sequences. The nucleic acid molecules of the inventioninclude full-length genes or cDNA molecules as well as a combination offragments thereof. The nucleic acids of the invention are derived fromhuman sources but the invention includes those derived from non-humanspecies.

An “isolated nucleic acid” is a nucleic acid that has been separatedfrom adjacent genetic sequences present in the genome of the organismfrom which the nucleic acid was isolated, in the case of nucleic acidsisolated from naturally-occurring sources. In the case of nucleic acidssynthesized enzymatically from a template or chemically, such as PCRproducts, cDNA molecules, or oligonucleotides for example, it isunderstood that the nucleic acids resulting from such processes areisolated nucleic acids. An isolated nucleic acid molecule refers to anucleic acid molecule in the form of a separate fragment or as acomponent of a larger nucleic acid construct. In one preferredembodiment, the nucleic acids are substantially free from contaminatingendogenous material. The nucleic acid molecule has preferably beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

Variant sequences can be prepared by site specific mutagenesis ofnucleotides in the DNA encoding the polypeptide, using cassette or PCRmutagenesis or other techniques well known in the art, to produce DNAencoding the variant, and thereafter expressing the recombinant DNA incell culture as outlined herein.

The present invention also provides expression systems and constructs inthe form of plasmids, expression vectors, transcription or expressioncassettes which comprise at least one polynucleotide as above. Inaddition, the invention provides host cells comprising such expressionsystems or constructs.

In one embodiment, the present invention provides a method of preparinga heterodimeric antibody or fragment thereof comprising an engineered VHand CH1 domain and an engineered VL and CL domain wherein the VH and CH1domains comprise a charged amino acid at positions 39, 147 and 165 (EUnumbering) and the VL and CL domains comprise a charged amino acid atpositions 38, 124 and 169/170 (EU numbering), the method comprising thesteps of: (a) culturing a host cell comprising a nucleic acid encodingthe engineered VH and CH1 domain polypeptides and a nucleic acidcomprising the engineered VL and CL domain polypeptides, wherein thecultured host cell expresses the engineered polypeptides; and (b)recovering the heterodimeric antibody from the host cell culture.

Expression vectors of use in the invention may be constructed from astarting vector such as a commercially available vector. After thevector has been constructed and a nucleic acid molecule encoding lightchain, a heavy chain, or a light chain and a heavy chain sequence hasbeen inserted into the proper site of the vector, the completed vectormay be inserted into a suitable host cell for amplification and/orpolypeptide expression. The transformation of an expression vector intoa selected host cell may be accomplished by well-known methods includingtransfection, infection, calcium phosphate co-precipitation,electroporation, microinjection, lipofection, DEAE-dextran mediatedtransfection, or other known techniques. The method selected will inpart be a function of the type of host cell to be used. These methodsand other suitable methods are well known to the skilled artisan, andare set forth, for example, in Sambrook et al., 2001, supra.

Typically, expression vectors used in the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas ‘flanking sequences’, in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element.

A host cell, when cultured under appropriate conditions, can be used toexpress bispecific antibody that can subsequently be collected from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity (such as glycosylation orphosphorylation) and ease of folding into a biologically activemolecule. A host cell may be eukaryotic or prokaryotic.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC) and any celllines used in an expression system known in the art can be used to makethe recombinant polypeptides of the invention. In general, host cellsare transformed with a recombinant expression vector that comprises DNAencoding a desired bispecific antibody. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 cells, L cells, C127 cells, 3T3 cells, Chinesehamster ovary (CHO) cells, or their derivatives and related cell lineswhich grow in serum free media, HeLa cells, BHK cell lines, the CVIIEBNAcell line, human embryonic kidney cells such as 293, 293 EBNA or MSR293, human epidermal A431 cells, human Colo205 cells, other transformedprimate cell lines, normal diploid cells, cell strains derived from invitro culture of primary tissue, primary explants, HL-60, U937, HaK orJurkat cells. Optionally, mammalian cell lines such as HepG2/3B, KB, NIH3T3 or S49, for example, can be used for expression of the polypeptidewhen it is desirable to use the polypeptide in various signaltransduction or reporter assays. Alternatively, it is possible toproduce the polypeptide in lower eukaryotes such as yeast or inprokaryotes such as bacteria. Suitable yeasts include S. cerevisiae, S.pombe, Kluyveromyces strains, Candida, or any yeast strain capable ofexpressing heterologous polypeptides. Suitable bacterial strains includeE. coli, B. subtilis, S. typhimurium, or any bacterial strain capable ofexpressing heterologous polypeptides. If the bispecific antibody is madein yeast or bacteria, it may be desirable to modify the product producedtherein, for example by phosphorylation or glycosylation of theappropriate sites, in order to obtain a functional product. Suchcovalent attachments can be accomplished using known chemical orenzymatic methods.

EXAMPLES Example 1: Rational Design

Rational design strategies were used to produce a set of proteins toidentify mutations that exhibit desired properties such as correct heavychain-light chain pairing. The design strategy for engineering of heavychain-light chain pairing included identifying a representative Fab. Acriterion for a representative Fab was that it was a member of commonlyused VH and VL subgroup such as VH3 and Vκ1, or lambda light chain.After the selection of a Fab (anti c-Kit heavy chain, SEQ ID NO: 7 andlight chain, SEQ ID NO: 21), an in silico analysis of the Fab interfacewas carried out to identify residues important for possible interactionsbetween heavy and light chains.

After introducing a set of mutations in the variable domains VH and VKand constant domains CH1 and CK a model was generated with the modelingsoftware MOE (Chemical Computing Group Inc.) using AMBER99 force-field.Blastp 2.2.30+ program was used to run the mutated domains against thePDB database. The pdb X-ray structure with the highest identity waschosen to model the best variant (pdb 3KDM).

1.1 Selection of Residue Positions

In order to ensure the correct pairing of light chain to its cognateheavy chain methods were investigated to control the heavy chain-lightchain assembly. The preferred approach was to control the assembly bycharge engineering of the VH-VL and CH1-CL domains. In this example,appropriate positions for charge engineering were selected byhuman-guided design and several criteria were applied. The chosen aminoacids should not be in contact with CDRs and, if possible, they shouldbe highly conserved within most common antibody families. Positions inthe centre of the interfaces should be avoided; rather they should be atthe rim of the interfaces to achieve a “clip-effect”. One arm shouldhave a set of charged residues and the other arm should have countercharges or a neutral charge at the corresponding 3D positions. Theresidues that met the criteria for engineering are listed below in Table1 and some of these were explored to make heterodimeric antibodies.Homology modelling was applied to check the distances of chosen sidechains after energy minimization. Models were generated with themodelling software MOE (Chemical Computing Group Inc.) using AMBER99force-field. Only side chain distances less than 10 Å were consideredfor further experiments. For example, the distance of K147D to T129R was10.1 Å and this pairing was not pursued further.

TABLE 1 HC kappa LC lambda LC EU number Residue EU number Residue EUnumber Residue Q39 R/K/D Q38 K/R/D Q38 K/R/D K147 K/D Q124 K/D E124E/K/D S165 R/D K169 K/D N170 K/D

Example 2: Expression and Purification of Engineered Fabs

Heavy chain and light chain DNA was synthesized at GeneArt (Regensburg,Germany) and cloned into a mammalian expression vector using restrictionenzyme-ligation based cloning techniques. The resulting plasmids wereco-transfected into HEK293T cells. For transient expression of Fabs,equal quantities of vector for each engineered chain were co-transfectedinto suspension-adapted HEK293T cells using Polyethylenimine (PEI;Cat#24765 Polysciences, Inc.). Typically, 100 ml of cells in suspensionat a density of 1-2 Mio cells per ml was transfected with DNA containing50 μg of expression vector encoding the engineered heavy chain and 50 μgexpression vectors encoding the engineered light chain. The recombinantexpression vectors were then introduced into the host cells and theconstruct produced by further culturing of the cells for a period of 7days to allow for secretion into the culture medium (HEK, serum-feemedium) supplemented with 0.1% pluronic acid, 4 mM glutamine, and 0.25μg/ml antibiotic. The produced construct was then purified fromcell-free supernatant using immunoaffinity chromatography. Filteredconditioned media was mixed with 300 μl Protein A resin (CaptivAPriMab™, Repligen), equilibrated with PBS buffer pH 7.4. The resin waswashed three times with 15 column volumes of PBS pH 7.4 before the Fabwas eluted with 10 column volumes protein A elution buffer (50 mMcitrate, 90 mM NaCl, pH 2.5).

For proof-of-concept studies a Fab of an anti c-Kit antibody generatedin-house, was selected. Competition studies were performed wherein twoengineered light chains had the option to bind two different engineeredheavy chains. To facilitate the validation, two additional alanineresidues were fused to the C-termini of certain light chains or heavychains, whereas other light chains and heavy chains were kept as chargeengineered entities only. These different charge engineered chains withor without the two alanine residues resulted in different masses asshown in Table 2.

TABLE 2 SEQ Mass SEQ Mass ID NO HC Fab* (MW-Da) ID NO LC Fab* (MW-Da) 1Q39K/K147D/S165R 24080.0 15 Q38D/Q124K/K169D 23379.1 2Q39K/K147D/S165R/CterAA 24222.2 16 Q38D/Q124K/K169D/CterAA 23521.2 3Q39K/K147D/S165 24010.9 17 Q38D/Q124K/K169 23392.1 4 Q39K/K147/S165R24093.1 18 Q38D/Q124/K169D 23379.0 5 Q39D/K147/S165D 24038.9 19Q38K/Q124D/K169 23392.1 6 Q39D/K147/S165D/CterAA 24181.1 20Q38K/Q124D/K169/CterAA 23534.3 7 Q39/K147/S165 24023.9 21 Q38/Q124/K16923405.1 8 Q39/K147/S165/CterAA 24166.1 22 Q38/Q124/K169/CterAA 23547.3 9Q39/K147D/S165R 24080.0 23 Q38/Q124K/K169D 23392.1 10 Q39K/K147/S16524024.0 24 Q38/Q124K/K169 23405.2 11 Q39/K147D/S165 24010.9 25Q38D/Q124/K169 23392.1 12 Q39/K147/S165R 24093.1 26 Q38/Q124/K169D23392.1 13 Q39D/K147/S165 24011.0 27 Q38K/Q124/K169 23405.2 14Q39/K147/S165D 24052.0 28 Q38/Q124D/K169 23392.1 *Q39K for example,indicates a mutation from Gln to Lys at position 39. Q39 for example,indicates no mutation was made at position 39.

Example 3: Mass Spectrometry Analysis of Engineered Fabs

Evaluation of correct heavy chain-light chain pairing of Fab variantswas done by liquid chromatography-mass spectrometry (LC-UV-ESI-MS). Inbrief, purified proteins were concentrated to 100 μL using spinconcentrators (3000 MWCO, Millipore) and analysed by reversed phasechromatography (column BEH C4 1.7 μm 2.1*50 mm, Waters) on an UPLC(Acquity UPLC I-Class, Waters) with 100% water, 0.05% Trifluoro aceticacid (TFA) and 100% acetonitrile, 0.04% TFA. Fab variants were separatedat 80° C. by applying a first gradient of 5%-10% of acetonitrile, 0.04%in 0.2 minutes, and a second gradient of 10% to 45% of acetonitrile,0.04% in 4 minutes. Eluted Fab variants were detected by UV (210-450 nm)and ionized by electrospray ionization (ESI) before analysis of theirmass by QTOF (Xevo G2-S QTof, Waters). Finally, the relative compositionof the injected Fab mixture was determined by double integration of UVsignal and mass intensity.

Results of the pairing of different HC-LC combinations are shown inTable 3. The right hand column shows the percentage of correct HC-LCpairing. A value of 100% indicates that there was no mispairing and 100%of light chain bound to its cognate heavy chain. A value of 95%, forexample, indicates that 95% of light chain bound to its cognate heavychain but there was 5% of mispairing where the light chain did not bindto its cognate heavy chain.

TABLE 3 HC LC Correct pairing (%) Q39K/K147D/S165R Q38D/Q124K/K169D 100Q39K/K147D Q38D/Q124K 95 Q39K/S165R Q38D/K169D 75 K147D/S165RQ124K/K169D 90 Q39K Q38D 74 K147D Q124K 91 S165R K169D 68Q39D/K147/S165D Q38K/Q124D/K169 100 Q39D/S165D Q38K/K169 95 Q39D/K147Q38K/Q124D 93 K147/S165D Q124D/K169 88 Q39D Q38K 95 S165D K169 87 K147Q124D 91 WT WT 63

Example 4: Translation to IgG with Two Kappa Light Chains(VH3-VK_VH3-VK)

Several preferred mutation-sets were introduced into the variable andconstant regions of a number of IgG1s. In addition to an anti c-Kitantibody (SEQ ID Nos: 29 and 30), anti-HER3 (SEQ ID NOs: 31 and 32) andanti-IL-17 (SEQ ID NO: 33 and 34) antibodies were used to evaluate theHC-LC pairing in an IgG1 setting. All three antibodies are members ofsubgroups VH3A/K1. For correct assembly of heavy chains the ‘knobs intoholes’ technology (Ridway et al., supra) was used with mutationsintroduced into the CH3 domain of the antibodies. Additional mutationswere introduced into the variable, CH1 and CL domains of the antibodiesaccording to Table 4 below.

TABLE 4 SEQ SEQ ID ID Antibody NO HC mutation NO LC mutation anti-HER331 parental 32 parental anti-IL-17 33 parental 34 parental anti-c-Kit 29parental 30 parental anti-HER3 (a) 37 Q39K/K147D/ 38 Q38D/Q124K/ S165RK169D anti-HER3 (b) 39 Q39D/K147/ 40 Q38K/Q124D/ S165D K169 anti-IL-1741 Q39K/K147D/ 42 Q38D/Q124K/ S165R K169D anti-c-Kit 43 Q39D/K147/ 44Q38K/Q124D/ S165D K169

Heterodimeric antibodies comprising the mutation sets were generatedusing standard transient HEK expression in the same cell and wereevaluated for correct assembly and biochemical and biophysicalproperties. Furthermore, the engineered heterodimeric antibodies wereevaluated for simultaneous binding of antigens. A comparison withparental, unmutated IgG1s is shown in Table 5. The percentage of Fabheterodimerization was determined by liquid chromatography-massspectrometry (LC-UV-ESI-MS; as described in Example 3). A value of 72%,for example, as observed for the IL-17/HER3 heterodimeric antibody,indicates that 72% of IL-17 and HER3 light chains bound to their cognateheavy chains but there was 28% of mispairing where the IL-17 light chainbound to the HER3 heavy chain and the HER3 light chain bound to theIL-17 heavy chain. A value of 100% indicates that there was nomispairing and 100% of light chains bound to their cognate heavy chains,as was observed for mutated IL-17/mutated HER3, and mutatedIL-17/mutated c-Kit and mutated c-Kit/mutated HER3 heterodimericantibodies.

TABLE 5 Simulta- neous Antibody 1/ Heterodimerization BIAcore ® - DSC-Tm binding of Antibody 2 of Fab (%) KD (nM) (° C.) antigens c-Kit/— —20    76.5 — HER3/— — 1   75.5 — IL-17/— — 0.01 79 — IL-17/HER3 72 — — —IL-17/c-Kit 73 — — — c-Kit/HER3 45 — — — Mutated 100 0.01/4  74.5/72.5yes IL-17/ Mutated HER3 (b) Mutated 100 0.07/20 74.5/72   yes IL-17/Mutated c-Kit Mutated 100  22/0.4   72/72.5 yes c-Kit/ Mutated HER3 (a)

Example 5: Translation to IgGs with One Kappa and One Lambda Light Chain(VH3-VK_VH3-VL)

To evaluate the effect of introducing charges into the Fab part of anantibody, antibodies with kappa light chains were mixed with antibodiescontaining lambda light chains. Preferred mutation-sets were introducedinto IgG1s. In addition to an anti-c-Kit antibody (SEQ ID NOs: 29 and30), anti-HER3 (SEQ ID NOs: 31 and 32), anti-IL-17 (SEQ ID NOs: 33 and34), an anti-IL-18 (SEQ ID NOs: 35 and 36) antibodies were used toevaluate the heavy chain-light chain pairing in an IgG1 setting. c-Kit,HER3, and IL-17 antibodies are members of the subgroups VH3-Vκ1, whereasthe IL-18 antibody contains a light chain which is a member of subgroupVL1. To ensure correct assembly of heavy chains the “knobs into holes”technology (supra) was used with mutations introduced into the CH3domain of the antibodies. Additional mutations were introduced into thevariable, CH1 and CL domains of the antibodies according to Table 6below.

TABLE 6 SEQ SEQ ID ID Antibody NO HC mutation NO LC mutation anti-HER331 parental 32 parental anti-IL-17 33 parental 34 parental anti-c-Kit 29parental 30 parental anti-IL-18 35 parental 36 parental anti-HER3 37Q39K/K147D/S165R 38 Q38D/Q124K/K169D anti-IL-17 41 Q39K/K147D/S165R 42Q38D/Q124K/K169D anti-c-Kit 45 Q39K/K147D/S165R 46 Q38D/Q124K/K169Danti-IL-18 47 Q39D/K147/S165D 48 Q38K/E124D/N170K

Heterodimeric antibodies comprising the mutation sets were generated andevaluated for correct assembly. A comparison with parental, unmutatedIgG1s is shown in Table 7. The right hand column shows the percentage ofFab heterodimerization as determined using liquid chromatography-massspectrometry (LC-UV-ESI-MS; as described in Example 3). A value of 71%as observed for the IL-17/IL-18 heterodimeric antibody indicates that71% of IL-17 and IL-18 light chains bound to their cognate heavy chainsbut there was 29% of mispairing where the IL-17 light chain bound to theIL-18 heavy chain and the IL-18 light chain bound to the IL-17 heavychain. A value of 100% indicates that there was no mispairing and 100%of light chains bound to their cognate heavy chains, as was observed formutated IL-17/mutated IL-18 and mutated c-Kit/mutated IL-18heterodimeric antibodies.

TABLE 7 Antibody 1/Antibody 2 Heterodimerization (%) of Fab IL-17/IL-1871 HER3/IL-18 20 c-Kit/IL-18 82 Mutated IL-17/Mutated IL-18 100 MutatedHER3/Mutated IL-18 95 Mutated c-Kit/Mutated IL-18 100

Example 6: Surface Plasmon Resonance (SPR) Binding Analysis

A direct binding assay was performed to characterize the binding of theengineered antibodies against their antigen. Kinetic binding affinityconstants (KD) were measured on protein-A captured protein usingrecombinant human antigens as analyte. Measurements were conducted on aBIAcore® T200 (GE Healthcare, Glattbrugg, Switzerland) at roomtemperature. For affinity measurements, the proteins were diluted in 10mM NaP, 150 mM NaCl, 0.05% Tween 20, pH5.8 and immobilized on the flowcells of a CM5 research grade sensor chip (GE Healthcare, refBR-1000-14) using standard amine coupling procedure according to themanufacturer's recommendation (GE Healthcare). To serve as reference,one flow cell was blank immobilized. Binding data were acquired bysubsequent injection of analyte dilutions series on the reference andmeasuring flow cell. Zero concentration samples (running buffer only)were included to allow double referencing during data evaluation. Fordata evaluation, doubled referenced sensograms were used and analyzed bysteady state analysis to generate the equilibrium dissociation constant(KD). The results are summarized in Table 5 (column BIAcore®-KD (nM).

Example 7: Differential Scanning Calorimetry (DSC)

The thermal stability of engineered heterodimeric antibodies and theirparental antibodies were compared using calorimetric measurements asshown in Table 5 (column DSC-Tm (° C.)). calorimetric measurements werecarried out on a differential scanning micro calorimeter (Nano DSC, TAinstruments). The cell volume was 0.5 ml and the heating rate was 1°C./min. All proteins were used at a concentration of 1 mg/ml in PBS (pH7.4). The molar heat capacity of each protein was estimated bycomparison with duplicate samples containing identical buffer from whichthe protein had been omitted. The partial molar heat capacities andmelting curves were analysed using standard procedure. Thermograms werebaseline corrected and concentration normalized.

Example 8: Translation of Charge Engineering into IgGs with DifferentFrameworks

To evaluate the effect of introducing charges into the Fab of antibodieswith different frameworks, antibodies with light chain frameworks Vκ1,Vκ3, VL1, VL2, VL3 and heavy chain frame works VH1, VH2, VH3, VH5 andVH6 were mixed with an antibody containing kappa light chain Vκ1 andheavy chain VH3. The sequences of framework regions of human origin maybe obtained from The Immunoglobulin Factsbook, by Marie-Paule Lefranc,Gerard Lefranc, Academic Press 2001, ISBN 012441351. Preferredmutation-sets were introduced into IgG1s and compared to theircorresponding non-engineered parental antibodies. Antibodies to HER2 andfive other antigens A-E, were combined with an anti-IL-17 antibody andused to evaluate the heavy chain-light chain mispairing in an IgG1setting. To ensure correct assembly of heavy chains the “knobs intoholes” technology (supra) was used.

Bispecific antibodies were generated having a first binding arm thattargeted the antigen HER2 or a number of other antigens listed here asantigens A-E, and a second binding arm that targeted IL-17. Engineeredantibodies to HER2 comprised the substitutions Q39D and S165D in the VHand CH1 domains and Q38K and Q124D in the kappa VL and CL domains orQ38K and N170R in the lambda VL and CL domains. Engineered antibodies toantigen A comprised the substitutions Q39D and S165D in the VH and CH1domains and Q38K or Q38K and N170R in the lambda VL and CL domains.Engineered antibodies to antigens B, D and E comprised the substitutionsQ39D and S165D in the VH and CH1 domains and Q38K and N170R in thelambda VL and CL domains. Engineered antibodies to antigen C comprisedthe substitutions Q39D and S165D in the VH and CH1 domains and Q38K inthe lambda VL domain. Engineered antibodies to IL-17 comprised thesubstitutions Q39K, K147D and S165R in the VH and CH1 domains and Q38D,Q124K and K169D in the kappa VL and CL domains. A comparison of heavychain-light chain pairing of the engineered bispecific antibodies withparental bispecific antibodies is shown in Table 8 below. For most ofthe engineered bispecific antibodies generated, mispairing, measured asa percentage of Fab heterodimerization by liquid chromatography-massspectrometry was fully eliminated. Mispairing was only observed for 3engineered bispecific antibodies up to a maximum of 5%, compared to therespective parental bispecific antibodies. This example clearly showsthat the incidence of heavy-light chain mispairing for bispecificheterodimeric antibodies can be almost eliminated by using the specificelectrostatic engineering mutations as disclosed herein and is notlimited to bispecific heterodimeric antibodies comprising specificframework regions.

Example 9: Translation of Charge Engineering Plus Disulfide BondEngineering into IgGs with Different Frameworks

The effect of replacing the native LC-HC interchain disulfide bond withan engineered VH-VL disulfide bond in one Fab of a bispecific antibodyin addition to introducing charges into the Fabs of the bispecificantibody, was evaluated. Antibodies with light chain frameworks VL2 orVL3 and heavy chain frameworks VH1, VH2, and VH5 were mixed with anantibody containing kappa light chain Vκ1 and heavy chain VH3. Thesequences of framework regions of human origin may be obtained from TheImmunoglobulin Factsbook (supra). Preferred electrostatic mutation-setswere introduced into IgG1s with the addition of an engineered VH-VLdisulfide bond into one Fab. These engineered antibodies with chargedresidues and a non-native disulfide bond in one Fab were compared to thecorresponding parental antibodies engineered with a non-native disulfidebond in one arm only. Antibodies to four antigens (antigens F, G, H andI) were combined with an anti-IL-17 antibody and used to evaluate theheavy chain-light chain mispairing in an IgG1 setting. To ensure correctassembly of heavy chains the “knobs into holes” technology (supra) wasused.

Bispecific antibodies were generated having a first binding arm thattargeted the antigens listed here as antigens F to I, and a secondbinding arm that targeted IL-17. Engineered antibodies to antigens F toI comprised the substitutions Q39D and S165D in the VH and CH1 domainsand Q38K and N170R in the lambda VL and CL domains. Engineeredantibodies to IL-17 (SEQ ID NOs: 73 and 74) comprised the substitutionsQ39K, G44C, K147D, S165R and C220A in the VH and CH1 domains and Q38D,Q100C, Q124K, K169D and C214A in the kappa VL and CL domains. Forcomparison, the anti-IL-17 antibody with only disulfide bond engineeringcomprised substitutions G44C and C220A in VH-CH1 domains and Q100C,C124A in the LC, respectively (SEQ ID NOs: 75 and 76). The outcome ofthe comparison is shown in FIG. 1 with details of the well content andengineered positions given in Table 9 below.

The analytical procedure for detecting and quantifying correctly pairedvs mispaired bispecific antibodies was greatly simplified using thismethod as only the correctly assembled bispecific antibodies couldmigrate as full length antibody (150 kDa) on a SDS-PAGE or any similarelectrophoresis based system. In the case of mispairing, where the lightchains and heavy chains were not covalently linked, incomplete moleculesand free light chain appeared on the gel. Mispairing was observed forbispecific antibodies generated without charge engineering, where free,unpaired light chain (25 kDa bands) and incomplete molecules (125 kDabands) were detected on the gel (FIG. 1; wells 7 to 10). In contrary,bispecific antibodies combining disulfide bond and charge engineeringshowed only traces of these bands (FIG. 1; wells 2 to 5). This exampleclearly shows that the incidence of heavy-light chain mispairing forbispecific heterodimeric antibodies can be eliminated by using specificelectrostatic engineering mutations as disclosed herein in combinationwith LC-HC interchain disulfide bond engineering in one Fab of thebispecific antibody. Disulfide bond engineering by itself appeared to benot sufficient to drive correct LC-HC pairing. In addition, theanalytical procedure is simplified since heavy-light chain mispairingcan be measured by electrophoresis, which enables high-through screeningof bispecific candidates with the correct assembly of their cognateheavy and light chains.

TABLE 8 Knob arm Hole arm SEQ ID HC 1* SEQ ID LC 1* Target HC 2 LC 2Target Mispairing (%) 54 VH3-23*01 49 Vk1-39*01 HER-2 VH3_7*01 Vk1_13*02IL-17 42 54 VH3-23*01 50 Vλ3-9*01 SEQ ID: 33 SEQ ID: 34 33 55 VH3-30*0151 Vk3-15*01 11 56 VH3-15*01 52 Vλ1-47*01 17 57 VH1-46*01 49 Vk1-39*0144 58 VH5-51*01 53 Vk1-33*01 74 59 VH6-1*01 52 Vλ1-47*01 39 VH1-2*02Vλ1-1-40*01 antigen A 30 VH2-70D*14 Vλ1-1-40*01 antigen B 59 VH3-23D*01Vλ2-14*01 antigen C 18 VH3-23D*01 Vλ3-1*01 antigen A 28 VH3-15*05Vλ1-1-40*01 antigen A 5 VH5-51*03 Vλ3-1*01 antigen D 69 VH6-1*02Vλ1-1-40*01 antigen E 79 VH6-1*02 Vλ2-14*01 antigen E 60 Engineered* HC1Engineered* LC1 Engineered HC2 Engineered LC2 67 VH3-23*01 62 Vk1-39*01HER2 VH3_7*01 Vk1_13*02 IL-17 0 Q39D/S165D Q38K/Q124D SEQ ID 61 SEQ ID60 67 VH3-23*01 63 Vλ3-9*01 Q39K/K147D/ Q38D/Q124K/ 0 Q39D/S165DQ38K/N170R S165R K169D 68 VH3-30*01 64 Vk3-15*01 0 Q39D/S165D Q38K/Q124D69 VH3-15*01 65 Vλ1-47*01 0 Q39D/S165D Q38K/N170R 70 VH1-46*01 62Vk1-39*01 0 Q39D/S165D Q38K/Q124D 71 VH5-51*01 66 Vk1-33*01 0 Q39D/S165DQ38K/Q124D 72 VH6-1*01 65 Vλ1-47*01 0 Q39D/S165D Q38K/N170R VH1-2*02Vλ1-1-40*01 antigen A 0 Q39D/S165D Q38K/N170R VH2-70D*14 Vλ1-1-40*01antigen B 0 Q39D/S165D Q38K/N170R VH3-23D*01 Vλ2-14*01 antigen C 0Q39D/S165D Q38K/N170R VH3-23D*01 Vλ3-1*01 antigen A 4 Q39D/S165DQ38K/N170R VH3-15*05 Vλ1-1-40*01 antigen A 0 Q39D/S165D Q38K/N170RVH5-51*03 Vλ3-1*01 antigen D 5 Q39D/S165D Q38K/N170R VH6-1*02Vλ1-1-40*01 antigen E 3 Q39D/S165D Q38K/N170R VH6-1*02 Vλ2-14*01 antigenE 0 Q39D/S165D Q38K/N170R *The sequences of framework regions of humanorigin may be obtained from The Immunoglobulin Factsbook, by Marie-PauleLefranc, Gerard Lefranc, Academic Press 2001, ISBN 012441351

TABLE 9 Knob arm Hole arm Wells HC 1 LC 1 Target HC2 LC2 Target 2VH1-2*02 Vλ3-1*01 antigen F VH3_7*01 Vk1_13*02 IL-17 Q39D/S165DQ38K/N170R SEQ ID 73 SEQ ID 74 3 VH2-70D*14 Vλ2-14*01 antigen GQ39K/G44C/K147D/ Q38D/Q100C/Q124K/ Q39D/S165D Q38K/N170R S165R/C220AK169D/C214A 4 VH5-51*03 Vλ2-14*01 antigen H Q39D/S165D Q38K/N170R 5VH5-51*03 Vλ3-1*01 antigen I Q39D/S165D Q38K/N170R 7 VH1-2*02 Vλ3-1*01antigen F VH3_7*01 Vk1_13*02 8 VH2-70D*14 Vλ2-14*01 antigen G SEQ ID 75SEQ ID 76 9 VH5-51*03 Vλ2-14*01 antigen H G44C/C220A Q100C/C214A 10VH5-51*03 Vλ3-1*01 antigen I 1, 6, 11 Molecular weight marker

TABLE 10 Sequence Table SEQ ID NO Description Sequence 1 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRKAPGK Q39K/K147D/S165RGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 2 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRKAPGK Q39K/K147D/S165R/GLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRA C-TER AAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCAA 3 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRKAPGK Q39K/K147D/S165GLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 4 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRKAPGK Q39K/K147/S165RGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 5 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRDAPGK Q39D/K147/S165DGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 6 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRDAPGK Q39D/K147/S165D/GLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRA CTERAAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCAA 7 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRQAPGK Q39/K147/S165GLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 8 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRQAPGK Q39/K147/S165/GLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRA CTERAAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCAA 9 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRQAPGK Q39/K147D/S165RGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 10 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRKAPGK Q39K/K147/S165GLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 11 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRQAPGK Q39/K147D/S165GLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 12 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRQAPGK Q39/K147/S165RGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 13 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRDAPGK Q39D/K147/S165GLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 14 ANTI-C-KIT HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRQAPGK Q39/K147/S165DGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSC 15 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQDKPGKAPK Q38D/Q124K/K169DLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSDDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 16 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQDKPGKAPK Q38D/Q124K/K169D/LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQG CTERAARRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSDDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAA 17 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQDKPGKAPK Q38D/Q124K/K169LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 18 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQDKPGKAPK Q38D/Q124/K169DLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSDDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 19 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQKKPGKAPK Q38K/Q124D/K169LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEDLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 20 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQKKPGKAPK Q38K/Q124D/K169/LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQG CTERAARRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEDLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAA 21 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK Q38/Q124/K169LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 22 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK Q38/Q124/K169/LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQG CTERAARRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAA 23 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK Q38/Q124K/K169DLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSDDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 24 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK Q38/Q124K/K169LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 25 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQDKPGKAPK Q38D/Q124/K169LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 26 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK Q38/Q124/K169DLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSDDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 27 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQKKPGKAPK Q38K/Q124/K169LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 28 ANTI-C-KIT LCDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK Q38/Q124D/K169LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEDLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 29 ANTI-C-Kit HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRQAPGK PARENTALGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 30ANTI-C-Kit LC DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK PARENTALLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31 ANTI-HER3 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK PARENTALGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLR (LALA)AEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 32 ANTI-HER3 LCDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAP PARENTALKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 33 ANTI-IL-17 HCEVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK PARENTALGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLR (LALA)AEDTAVYYCARDRGSLYYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 34 ANTI-IL-17 LCAIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQQKPGKAPK PARENTALLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 35 ANTI-IL-18 HCEVQLVQSGAEVKKPGSSVKVSCKASGGTFKSYAISWVRQAPGQ PARENTALGLEWMGNIIPMTGQTYYAQKFQGRVTITADESTSTAYMELSSLRS (LALA)EDTAVYYCARAAYHPLVFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 36 ANTI-IL-18 LCDIVLTQPPSVSGAPGQRVTISCSGSSSNIGNHYVNWYQQLPGTAP PARENTALKLLIYRNNHRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSWDYSGFSTVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 37 ANTI-HER3 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRKAPGK Q39K/K147D/S165RGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLR (LALA)AEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 38 ANTI-HER3 LCDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQDKPGKAP Q38D/Q124K/K169DKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSDDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 39 ANTI-HER3 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRDAPGK Q39D/K147/S165DGLEWVSVTGAVGRSTYYPDSVKGRFTISRDNSKNTLYLQMNSLR (LALA)AEDTAVYYCARWGDEGFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 40 ANTI HER3 LCDIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQKKPGKAP Q38K/Q124D/K169KLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSFPTTFGQGTKVEIKRTVAAPSVFIFPPSDEDLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 41 ANTI-IL-17 HCEVQLVESGGDLVQPGGSLRSLCAASGFTFSSYWMSWVRKAPGK Q39K/K147D/S165RGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLR (LALA)AEDTAVYYCARDRGSLYYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 42 ANTI-IL-17 LCAIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQDKPGKAPK Q38D/Q124K/K169DLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSDDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 ANTI c-Kit HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRDAPGK Q39D/K147/S165DGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 44ANTI C-KIT LC DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQKKPGKAPKQ38K/Q124D/K169 LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEDLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 45 ANTI c-Kit HCEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRKAPGK Q39K/K147D/S165RGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 46ANTI C-KIT LC DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQDKPGKAPKQ38D/Q124K/K169D LLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSDDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 47 ANTI-IL-18 HCEVQLVQSGAEVKKPGSSVKVSCKASGGTFKSYAISVWRDAPGQG Q39D/K147/S165DLEWMGNIIPMTGQTYYAQKFQGRVTITADESTSTAYMELSSLRSE (LALA)DTAVYYCARAAYHPLVFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 48 ANTI-IL-18 LCDIVLTQPPSVSGAPGQRVTISCSGSSSNIGNHYVNWYQKLPGTAP Q38K/E124D/N170KKLLIYRNNHRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCQSWDYSGFSTVFGGGTKLTVLGQPKAAPSVTLFPPSSEDLQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSKNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 49 VK1_39*01DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK ANTI-HER2 LCLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKSDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 50 Vλ3_9*01SYELTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPV ANTI-HER2 LCLVIYRDSNRPSGIPERFSGSNSGNTATLTISRAQDEADYYCQVWDSSTVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 51 VK3_15*01EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAP ANTI-HER2 LCRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 52 Vλ1_47*01QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTA ANTI-HER2 LCPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 53 VK1_33*01DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK ANTI-HER2 LCLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 54 ANTI-HER2 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK VH3_23*01GLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 55 ANTI-HER2 HCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK VH3_30*01GLEWVAVISYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 56 ANTI-HER2 HCEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGK VH3_15*01GLEWVGRIKSKTEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 57 ANTI-HER2 HCQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQ VH1_46*01GLEWMGIISPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 58 ANTI-HER2 HCEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKG VH5_51*01LEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARWGGDGFYAMDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 59 ANTI-HER2 HCQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSSSAAWNWIRQSPS VH6_1*01RGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 60 ENGINEEREDAIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQDKPGKAPK VK1_13*02LLIYDASSLEQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQF ANTI-IL-17 LCNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEKLKSGTASVVCLLN Q38D/Q124K/K169DNFYPREAKVQWKVDNALQSGNSQESVTEQDSDDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 61 ENGINEEREDEVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRKAPGK VH3_7*01GLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLR ANTI-IL-17 HCAEDTAVYYCARDRGSLYYWGQGTLVTVSSASTKGPSVFPLAPSS Q39K/K147D/S165RKSTSGGTAALGCLVDDYFPEPVTVSWNSGALTRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 62 ENGINEEREDDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQKKPGKAPK VK1_39*01LLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQS ANTI-HER2 LCYSTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEDLKSGTASVVCLLN Q38K/Q124DNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 63 ENGINEEREDSYELTQPLSVSVALGQTARITCGGNNIGSKNVHWYQKKPGQAPV Vλ3_9*01LVIYDSNRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQV ANTI-HER2 LCWDSSTVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVC Q38K/N170RLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSRNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 64 ENGINEEREDEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQKKPGQAP VK3_15*01RLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQ ANTI-HER2 LCYNNWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEDLKSGTASVVCLL Q38K/Q124DNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 65 ENGINEEREDQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQKLPGTA Vλ1_47*01PKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC ANTI-HER2 LCAAWDDSLSGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANK Q38K/N170RATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSRNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 66 ENGINEEREDDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQKKPGKAPK VK1_33*01LLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQY ANTI-HER2 LCDNLPLTFGQGTKVEIKRTVAAPSVFIFPPSDEDLKSGTASVVCLLN Q38K/Q124DNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 67 ENGINEEREDEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRDAPGK ANTI-HER2 HCGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRA VH3_23*01EDTAVYYCARWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPL Q39D/S165DAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 68 ENGINEEREDEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRDAPGK ANTI-HER2 HCGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRA VH3_30*01EDTAVYYCARWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPL Q39D/S165DAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 69 ENGINEEREDEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRDAPGK ANTI-HER2 HCGLEVWGRIKSKTEGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSL VH3_15*01KTEDTAVYYCARWGGDGFYAMDYWGQGTLVTVSSASTKGPSVF Q39D/S165DPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 70 ENGINEEREDQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHVWRDAPGQ ANTI-HER2 HCGLEWMGIISPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLR VH1_46*01SEDTAVYYCARWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFP Q39D/S165DLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 71 ENGINEEREDEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRDMPGKG ANTI-HER2 HCLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKAS VH5_51*01DTAMYYCARWGGDGFYAMDYWGRGTLVTVSSASTKGPSVFPLA Q39D/S165DPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 72 ENGINEEREDQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSSSAAWNWIRDSPS ANTI-HER2 HCRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSV VH6_1*01TPEDTAVYYCARWGGDGFYAMDYWGQGTLVTVSSASTKGPSVF Q39D/S165DPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 73 ENGINEEREDEVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRKAPGK ANTI-IL-17 HCCLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRA Q39K/G44C/K147D/EDTAVYYCARDRGSLYYWGQGTLVTVSSASTKGPSVFPLAPSSK S165R/C220ASTSGGTAALGCLVDDYFPEPVTVSWNSGALTRGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 74 ENGINEEREDAIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQDKPGKAPK ANTI-IL-17 LCLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQF Q38D/Q100C/Q124K/NSYPLTFGCGTKVEIKRTVAAPSVFIFPPSDEKLKSGTASVVCLLN K169D/C214ANFYPREAKVQWKVDNALQSGNSQESVTEQDSDDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEA 75 ENGINEEREDEVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK ANTI-IL-17 HCCLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRA G44C/C220AEDTAVYYCARDRGSLYYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 76 ENGINEEREDAIQLTQSPSSLSASVGDRVTITCRPSQGINWELAWYQQKPGKAPK ANTI-IL-17 LCLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQF Q100C/C214ANSYPLTFGCGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEA

1. A heterodimeric antibody or fragment thereof comprising at least twoFabs, wherein at least one Fab comprises: (a) an engineered heavy chainvariable domain (VH) and constant domain 1 (CH1) comprising an acidicamino acid at position 39, a basic amino acid at position 147 and anacidic amino acid at position 165 (EU numbering); and an engineeredlight chain variable domain (VL) and constant domain (CL) comprising abasic amino acid at position 38, an acidic amino acid at position 124and a basic amino acid at positions 169/170 (EU numbering), or (b) anengineered VH domain and CH1 domain comprising a basic amino acid atposition 39, an acidic amino acid at position 147 and a basic amino acidat position 165 and an engineered VL domain and CL domain comprising anacidic amino acid at position 38, a basic amino acid at position 124 andan acidic amino acid at position 169/170, wherein said amino acids inthe VH and CH1 domains and in the VL and CL domains pairwise are ofopposing charge, and correspond to form an interface that iselectrostatically favourable to heterodimerization.
 2. The heterodimericantibody or fragment thereof of claim 1, wherein heterodimerization ofat least 95% is achieved, as determined by liquid chromatography-massspectrometry. 3.-4. (canceled)
 5. The heterodimeric antibody or fragmentthereof of claim 1, wherein the engineered heavy chain variable domainis of the VH1, VH2, VH3, VH5 or VH6 subtype.
 6. The heterodimericantibody or fragment thereof of claim 1, wherein the engineered lightchain variable domain is of the Vκ1 or Vκ3 subfamily and comprises acharged amino acid at positions 38, 124 and
 169. 7. The heterodimericantibody or fragment thereof of claim 1, wherein the engineered lightchain variable domain is of the Vλ1, Vλ2 or Vλ3 subfamily and comprisesa charged amino acid at positions 38, 124 and
 170. 8. The heterodimericantibody or fragment thereof of claim 1, comprising an engineered lightchain variable domain of the Vκ1 or Vκ3 subfamily and an engineeredlight chain variable domain of the Vλ1, Vλ2 or Vλ3 subfamily.
 9. Theheterodimeric antibody or fragment thereof of claim 1, comprising an IgGFc region.
 10. The heterodimeric antibody or fragment thereof of claim9, wherein the IgG Fc region comprises a first CH3 region having asubstitution at position 336 and a second CH3 region having asubstitution at positions 336, 368 and
 407. 11. The heterodimericantibody or fragment thereof of claim 9, wherein the IgG Fc regioncomprises a first CH3 region having a substitution at positions 336 and354 and a second CH3 region having a substitution at positions 336, 368,407 and
 349. 12. The heterodimeric antibody or fragment thereof of claim11, wherein the first CH3 region comprises the substitutions T366W andS354C and the second CH3 region comprises the substitutions T366S,L368A, Y407A and Y349C.
 13. The heterodimeric antibody or fragmentthereof of claim 1, comprising substitutions selected from the groupconsisting of: i. Q39D and S165D in the VH and CH1, and Q38K in the VL;ii. Q39K and K147D in the VH and CH1, and Q38D and Q124K in the kappa VLand CL; iii. Q39D and S165D in the VH and CH1, and Q38K and Q124D in thekappa VL and CL; iv. Q39K, K147D and S165R in the VH and CH1, and Q38D,Q124K and K169D in the kappa VL and CL; v. Q39D and S165D in the VH andCH1, and Q38K, E124D and N170K in the lambda VL and CL; and vi. Q39D andS165D in the VH and CH1, and Q38K and N170R in the lambda VL and CL. 14.The heterodimeric antibody or fragment thereof of claim 1, whereinheterodimerization of up to 100% is achieved, as determined by liquidchromatography-mass spectrometry.
 15. A heterodimeric antibody orfragment thereof of claim 1, wherein an additional Fab comprises: anengineered heavy chain variable domain (VH) and constant domain 1 (CH1)comprising a charged amino acid at positions 39, 147 and a charged orneutral amino acid at position 165 (EU numbering); and an engineeredlight chain variable domain (VL) and constant domain (CL) comprising acharged amino acid at positions 38 and a charged or neutral amino acidat positions 124 and 169/170 (EU numbering), wherein said amino acids inthe VH and CH1 domains and in the VL and CL domains pairwise are ofopposing charge or are charged/neutral, and correspond to form aninterface that is electrostatically favourable to heterodimerization;and wherein the charges at each of positions 39, 147 and 165 in theengineered VH of one Fab are different to the charges at each ofpositions 39, 147 and 165 in the engineered VH of the additional Fab.16. The heterodimeric antibody or fragment thereof of claim 15, whereinone Fab comprises the substitutions Q39K, K147D and S165R in the VH andCH1 and the substitutions Q38D, Q124K and K169D in the VL and CL; andthe additional Fab comprises the substitutions Q39D and S165D in the VHand CH1 and substitutions in the VL and CL selected from (i) Q38K andQ124D in the kappa VL and CL; (ii) Q38K in the lambda VL; (iii) Q38K andN170R in the lambda VL and CL; or (iv) Q38K, E124D and N170K in thelambda VL and CL.
 17. The heterodimeric antibody or fragment thereof ofclaim 15, wherein an interchain disulfide bond in the additional Fab isreplaced with an engineered VH-VL interchain disulfide bond.
 18. Theheterodimeric antibody or fragment thereof of claim 17, wherein one Fabcomprises the substitutions Q39K, K147D and S165R in the VH and CH1 andthe substitutions Q38D, Q124K and K169D in the VL and CL; and theadditional Fab comprises the substitutions Q39D, G44C, S165D and C220Ain the VH and CH1 and substitutions in the VL and CL selected from (i)Q38K, Q100C, Q124D and C214A in the kappa VL and CL; (ii) Q38K, G100Cand C214A in the lambda VL; (iii) Q38K, G100C N170R and C214A in thelambda VL and CL; or (iv) Q38K, G100C, E124D, N170K and C214A in thelambda VL and CL.
 19. A method of preparing an heterodimeric antibody orfragment thereof comprising an engineered VH and CH1 domain and anengineered VL and CL domain that correspond to form an interface, themethod comprising substituting at least two amino acids in the VH andCH1 domains such that the engineered domains comprise an acidic aminoacid at position 39, a basic amino acid at position 147 and an acidicamino acid at position 165 (EU numbering), and substituting an aminoacid in the VL and CL domains such that the engineered domains comprisea basic amino acid at position 38, an acidic amino acid at position 124and a basic amino acid at position 169/170 (EU numbering), and whereinsaid charged amino acids in the VH and CH1 domains and the correspondingamino acids of the interface in the VL and CL domains pairwise are ofopposing charge, and form an interface that is electrostaticallyfavourable to heterodimerization.
 20. The method of claim 19, whereinheterodimerization of at least 95% is achieved as determined by liquidchromatography-mass spectrometry. 21.-22. (canceled)
 23. The method ofclaim 19, wherein the engineered heavy chain variable domain is of theVH1, VH2, VH3, VH5 or VH6 subtype.
 24. The method of claim 19, whereinthe engineered light chain variable domain is of the Vκ1 or Vκ3subfamily and comprises a charged amino acid at positions 38, 124 and169.
 25. The method of claim 19, wherein the engineered light chainvariable domain is of the Vλ1, Vλ2 or Vλ3 subfamily and comprises acharged amino acid at positions 38, 124 and
 170. 26. The method of claim19, wherein the heterodimeric antibody or fragment thereof comprises anengineered light chain variable domain of the Vκ1 or Vκ3 subfamily andan engineered light chain variable domain of the Vλ1, Vλ2 or Vλ3subfamily.
 27. The method of claim 19, wherein the heterodimericantibody or fragment thereof comprising an IgG Fc region.
 28. The methodof claim 27, wherein the IgG Fc region comprises a first CH3 regionhaving a substitution at position 336 and a second CH3 region having asubstitution at positions 336, 368 and
 407. 29. The method of claim 27,wherein the IgG Fc region comprises a first CH3 region having asubstitution at positions 336 and 354 and a second CH3 region having asubstitution at positions 336, 368, 407 and
 349. 30. The method of claim29, wherein the first CH3 region comprises the substitutions T366W andS354C and the second CH3 region comprises the substitutions T366S,L368A, Y407A and Y349C.
 31. The method of claim 19, wherein theheterodimeric antibody or fragment thereof comprises the substitutionsselected from the group consisting of: i. Q39D and S165D in the VH andCH1, and Q38K the VL and CL; ii. Q39K and K147D in the VH and CH1, andQ38D and Q124K in the kappa VL and CL; and iii. Q39D and S165D in the VHand CH1, and Q38K and Q124D in the kappa VL and CL; iv. Q39K, K147D andS165R in the VH and CH1, and Q38D, Q124K and K169D in the kappa VL andCL; v. Q39D and S165D in the VH and CH1, and Q38K, E124D and N170K inthe lambda VL and CL; and vi. Q39D and S165D in the VH and CH1, and Q38Kand N170R in the lambda VL and CL.
 32. The method of claim 19, whereinheterodimerization of up to 100% is achieved as determined by liquidchromatography-mass spectrometry.
 33. The method of claim 19, whereinthe heterodimeric antibody or fragment thereof comprises an additionalFab comprising: an engineered heavy chain variable domain (VH) andconstant domain 1 (CH1) comprising a charged amino acid at positions 39,147 and a charged or neutral amino acid at position 165 (EU numbering);and an engineered light chain variable domain (VL) and constant domain(CL) comprising a charged amino acid at positions 38 and a charged orneutral amino acid at positions 124 and 169/170 (EU numbering), whereinsaid amino acids in the VH and CH1 domains and in the VL and CL domainspairwise are of opposing charge or are charged/neutral, and correspondto form an interface that is electrostatically favourable toheterodimerization; and wherein the charges at each of positions 39, 147and 165 in the engineered VH of one Fab are different to the charges ateach of positions 39, 147 and 165 in the engineered VH of the additionalFab.
 34. The method of claim 33, wherein one Fab comprises thesubstitutions Q39K, K147D and S165R in the VH and CH1 and thesubstitutions Q38D, Q124K and K169D in the VL and CL; and the additionalFab comprises the substitutions Q39D and S165D in the VH and CH1 andsubstitutions in the VL and CL selected from (i) Q38K and Q124D in thekappa VL and CL; (ii) Q38K in the lambda VL; (iii) Q38K and N170R in thelambda VL and CL; or (iv) Q38K, E124D and N170K in the lambda VL and CL.35. The method of claim 33, wherein a native LC-HC interchain disulfidebond of the additional Fab is replaced with an engineered VH-VLinterchain disulfide bond.
 36. The method 35 wherein one Fab comprisesthe substitutions Q39K, K147D and S165R in the VH and CH1 and thesubstitutions Q38D, Q124K and K169D in the VL and CL; and the additionalFab comprises the substitutions Q39D, G44C, S165D and C220A in the VHand CH1 and substitutions in the VL and CL selected from (i) Q38K,Q100C, Q124D and C214A in the kappa VL and CL; (ii) Q38K, G100C andC214A in the lambda VL; (iii) Q38K, G100C N170R and C214A in the lambdaVL and CL; or (iv) Q38K, G100C, E124D, N170K and C214A in the lambda VLand CL.
 37. A method of preparing a heterodimeric antibody or fragmentthereof according to claim 1, the method comprising the steps of: (a)culturing a host cell comprising a nucleic acid encoding the engineeredVH and CH1 domain polypeptides and a nucleic acid comprising theengineered VL and CL domain polypeptides, wherein the cultured host cellexpresses the engineered polypeptides; and (b) recovering theheterodimeric antibody or fragment thereof from the host cell culture.38. A method of preparing a heterodimeric antibody or fragment thereofaccording to claim 17, the method comprising the steps of: (a) culturinga host cell comprising a nucleic acid encoding the engineered VH and CH1domain polypeptides and a nucleic acid comprising the engineered VL andCL domain polypeptides, wherein the cultured host cell expresses theengineered polypeptides; (b) recovering the heterodimeric antibody orfragment thereof from the host cell culture; and (c) determining correctheavy chain-light chain pairing using an electrophoresis system.